The present invention relates to methods for sterilizing preparations of monoclonal immunoglobulins to reduce the level of active biological contaminants therein, such as viruses, bacteria, yeasts, molds, mycoplasmas, prions and/or parasites.
Antibodies are produced by organisms in response to exposure to foreign substances that the body perceives as a threat. Antibodies, or as they are collectively known, immunoglobulins (Ig), are proteins secreted by cells of the immune system known as B-cells or plasma cells. The structure of immunoglobulins is complex, but is well characterized. In brief, each immunoglobulin consists of a complex of protein chains known as the heavy and light chains. Each heavy chain is linked to a single light chain via disulfide bonds. The resulting complex is in turn linked by additional disulfide bonds to an identical heavy-light chain complex. This basic unit can be assembled by the cell into several specialized forms by varying the structure and number of heavy chains. Different heavy chain structures produce differing molecules, known as xe2x80x9cclassesxe2x80x9d of immunoglobulins. These classes may also have different numbers of the basic units described above.
The production of these various physical forms of the immunoglobulin molecule occurs in a sequential manner. During this process, the specificity of the molecule for a single molecule or antigen remains unchanged. This is because the changes described above all occur to the portion of the immunoglobulin molecule that is not involved in determining the specificity of the particular immunoglobulin molecule. This xe2x80x9chypervariable regionxe2x80x9d is subject to an unusually high degree of recombination events during B-cell maturation. These recombination events cease prior to the production of the first immunoglobulin molecule by the cell. The result is that from a relatively small number of variable region genes, the body generates a large number of potential immunoglobulin molecules of differing specificities.
Once a B-cell encounters a molecule to which its own immunoglobulin molecule binds (an xe2x80x9cantigenxe2x80x9d), and upon receiving signals from other cells in the immune system, the B-cell first multiplies into a large number of identical cells, (collectively referred to as a clone) and then differentiates into an immunoglobulin-secreting plasma cell. In this way, the extremely large number of potential immunoglobulin molecules that might be manufactured is limited to only those molecules that recognize antigens to which the body must respond.
The vast array of immunoglobulin specificities that are produced results in an ongoing protection for the body against infection from those organisms that the body has made immunoglobulins against in the past. Taken in sum, the result is that the immunoglobulins contained in the plasma from a single donor may have millions of useful immunoglobulin specificities. A preparation of immunoglobulins from plasma is thus referred to as a polyclonal immunoglobulin preparation, since it contains the immunoglobulin molecules produced by all of the plasma cell clones in the body.
Polyclonal immunoglobulins are particularly useful for treating human disease in which the ability to produce Ig is absent or impaired. Since all plasma cell clones are affected, a mixture of all the immunoglobulin specificities found in the plasma is needed to correct the deficiency. In contrast, when an extreme degree of specificity is required, or when a single defined therapeutic goal is sought, polyclonal immunoglobulins are not the best solution. Instead, an immunoglobulin preparation consisting of the immunoglobulin molecules produced by a single clone with the desired specificity is the most precise and predictable solution. Such a preparation is known as a monoclonal immunoglobulin.
Monoclonal immunoglobulin have many differences as compared to polyclonal immunoglobulins. Their monospecificity makes them very precise when used as detection reagents. As therapeutics, they are free of confounding or dangerous side effects that arise from polyclonal immunoglobulin preparations, such as the introduction of immunoglobulins of unwanted specificities being introduced into the patient. Their physical characteristics may also be different. Since each monoclonal immunoglobulin has a unique and unvarying structure, its potential for stability, degradation, aggregation, temperature sensitivity and other characteristics are unique and unchanging. Once a suitable monoclonal immunoglobulin has been chosen for production, its characteristics will not change, and it thus can be manufactured with great consistency and assurance of its performance and storage characteristics. The ability to tailor production volumes to product requirements also makes monoclonal immunoglobulin a highly desirable alternative to polyclonal immunoglobulins.
Monoclonal immunoglobulin preparations are made in one of three general fashions. The first involves production in a cell culture system, the second uses an animal as a temporary bioreactor for monoclonal immunoglobulin production, and the third involves inserting the gene for a desired monoclonal immunoglobulin into an animal in such a manner as to induce continuous production of the monoclonal immunoglobulin into a fluid or tissue of the animal so that it can be continuously harvested (transgenic production).
Each of these methods may result in contamination of the product by pathogens. In the first method, the cells producing the monoclonal immunoglobulin may harbour undetected viruses that can be produced in the culture system. Contamination of the culture system by bacteria, yeast or mold may also occur.
Both of the remaining methods involve the use of an animal to either serve as a host for the monoclonal immunoglobulin-producing cells or as a bioreactor to manufacture the monoclonal immunoglobulin product itself. Obviously, these products face the risk of contamination by pathogens infecting or harboured by the host animal. Such pathogens include, viruses, bacteria, yeasts, molds, mycoplasmas, and parasites, among others.
Consequently, it is of utmost importance that any biologically active contaminant in the monoclonal immunoglobulin product be inactivated before the product is used. This is especially critical when the product is to be administered directly to a patient. This is also critical for various monoclonal immunoglobulin products which are prepared in media which contain various types of plasma and which may be subject to mycoplasma or other viral contaminants.
Previously, most procedures have involved methods that screen or test products for a particular contaminant rather than removal or inactivation of the contaminant from the product. Products that test positive for a contaminant are merely not used. Examples of screening procedures include the testing for a particular virus in human blood from blood donors. Such procedures, however, are not always reliable and are not able to detect the presence of viruses in very low numbers. This reduces the value or certainty of the test in view of the consequences associated with a false negative result. False negative results can be life threatening in certain cases, for example in the case of Acquired Immune Deficiency Syndrome (AIDS). Furthermore, in some instances it can take weeks, if not months, to determine whether or not the product is contaminated.
In conducting experiments to determine the ability of technologies to inactivate viruses, the actual viruses of concern are seldom utilized. This is a result of safety concerns for the workers conducting the tests, and the difficulty and expense associated with the containment facilities and waste disposal. In their place, model viruses of the same family and class are used.
In general, it is acknowledged that the most difficult viruses to inactivate are those with an outer shell made up of proteins, and that among these, the most difficult to inactivate are those of the smallest size. This has been shown to be true for gamma irradiation and most other forms of radiation as these viruses diminutive size is a consequence of their small genome. The magnitude of direct effects of radiation upon a molecule are directly proportional to the size of the molecule, that is the larger the target molecule, the greater the effect. As a corollary, it has been shown for gamma-irradiation that the smaller the viral genome, the higher the radiation dose required to inactive it.
Among the viruses of concern for both human and animal-derived biologics, the smallest viruses of concern belong to the family of Parvoviruses and the slightly larger protein-coated Hepatitis virus. In humans, the Parvovirus B19, and Hepatitis A are the agents of concern. In porcine-derived products and tissues, the smallest corresponding virus is Porcine Parvovirus. Since this virus is harmless to humans, it is frequently chosen as a model virus for the human B19 Parvovirus and Hepatitis A. The demonstration of inactivation of this model parvovirus is considered adequate proof that the method employed will kill human B19 virus and Hepatitis A, and by extension, that it will also kill the larger and less hardy viruses such as HIV, CMV, Hepatitis B and C and others.
More recent efforts have focused on methods to remove or inactivate contaminants in the products. Such methods include heat treating, filtration and the addition of chemical inactivants or sensitizers to the product. Heat treatment requires that the product be heated to approximately 60xc2x0 C. for about 70 hours which can be damaging to sensitive products. Heat inactivation can destroy 50% or more of the biological activity of the product. Filtration involves filtering the product in order to physically remove contaminants. Unfortunately this method may also remove products that have a high molecular weight. Further, in certain cases small viruses may not be removed by the filter because of the larger molecular structure of the product. The procedure of chemical sensitization involves the addition of noxious agents which bind to the DNA/RNA of the virus and which are activated either by UV or radiation to produce reactive intermediates and/or free radicals which bind to the DNA/RNA or break the chemical bonds in the backbone of the DNA/RNA of the virus or crosslink or complex it in such a way that the virus can no longer replicate. This procedure requires that unbound sensitizer is washed from products since the sensitizers are toxic, if not mutagenic or carcinogenic, and can not be administered to a patient.
Irradiating a product with gamma radiation is another method of sterilizing a product. Gamma radiation is effective in destroying viruses and bacteria when given in high total doses (Keathly et al., xe2x80x9cIs There Life After Irradiation? Part 2,xe2x80x9d BioPharm July-August, 1993, and Leitman, Use of Blood Cell Irradiation in the Prevention of Post Transfusion Graft-vs-Host Disease,xe2x80x9d Transfusion Science 10:219-239 (1989)). The published literature in this area, however, teaches that gamma radiation can be damaging to radiation sensitive products, such as blood, blood products, protein and protein-containing products. In particular, it has been shown that high radiation doses are injurious to red cells, platelets and granulocytes (Leitman). U.S. Pat. No. 4,620,908 discloses that protein products must be frozen prior to irradiation in order to maintain the viability of the protein product. This patent concludes that xe2x80x9c[i]f the gamma irradiation were applied while the protein material was at, for example, ambient temperature, the material would be also completely destroyed, that is the activity of the material would be rendered so low as to be virtually ineffectivexe2x80x9d. This would apply as well to monoclonal immunoglobulins which are, of course, proteins. Unfortunately, many sensitive biologicals, such as monoclonal antibodies (Mab), would lose viability and activity if subjected to freezing for irradiation purposes and then thawing prior to administration to a patient.
In view of the difficulties discussed above, there remains a need for methods of sterilizing monoclonal immunoglobulins that are effective for reducing the level of active biological contaminants without an adverse effect on the monoclonal immunoglobulins.
Accordingly, it is an object of the present invention to provide methods of sterilizing preparations of monoclonal immunoglobulins by reducing the level of active biological contaminants without adversely affecting the monoclonal immunoglobulins. Other objects, features and advantages of the present invention will be set forth in the detailed description of preferred embodiments that follows, and in part will be apparent from the description or may be learned by practice of the invention. These objects and advantages of the invention will be realized and attained by the compositions and methods particularly pointed out in the written description and claims hereof.
In accordance with these and other objects, a first embodiment of the present invention is directed to a method for sterilizing a preparation of monoclonal immunoglobulins that is sensitive to radiation comprising: (i) reducing the residual solvent content of a preparation of monoclonal immunoglobulins to a level effective to protect the preparation of monoclonal immunoglobulins from radiation; and (ii) irradiating the preparation of monoclonal immunoglobulins with radiation at an effective rate for a time effective to sterilize the preparation of monoclonal immunoglobulins.
A second embodiment of the present invention is directed to a method for sterilizing a preparation of monoclonal immunoglobulins that is sensitive to radiation comprising: (i) adding to a preparation of monoclonal immunoglobulins at least one stabilizer in an amount effective to protect the preparation of monoclonal immunoglobulins from radiation; and (ii) irradiating the preparation of monoclonal immunoglobulins with radiation at an effective rate for a time effective to sterilize the preparation of monoclonal immunoglobulins.
A third embodiment of the present invention is directed to a method for sterilizing a preparation of monoclonal immunoglobulins that is sensitive to radiation comprising: (i) reducing the residual solvent content of a preparation of monoclonal immunoglobulins to a level effective to protect the preparation of monoclonal immunoglobulins from radiation; (ii) adding to the preparation of monoclonal immunoglobulins at least one stabilizer in an amount effective to protect the preparation of monoclonal immunoglobulins from radiation; and (iii) irradiating the preparation of monoclonal immunoglobulins with radiation at an effective rate for a time effective to sterilize the preparation of monoclonal immunoglobulins. According to this embodiment, steps (i) and (ii) may be reversed.
The invention also provides a composition comprising at least one monoclonal immunoglobulin and a least one stabilizer selected from the group consisting of: ascorbic acid or a salt or ester thereof; glutathione; 6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid; uric acid or a salt or ester thereof; methionine; histidine; N-acetyl cysteine; the dipeptide glycine-glycine; diosmin; silymarin; a mixture of ascorbic acid, or a salt or ester thereof, and uric acid, or a salt or ester thereof; a mixture of ascorbic acid, or a salt or ester thereof, and 6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid; a mixture of ascorbic acid, or a salt or ester thereof, uric acid, or a salt or ester thereof, and 6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid; and a mixture of uric acid, or a salt or ester thereof and 6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid, said at least one stabilizer being present in an amount effective to preserve said monoclonal immunoglobulin for its intended use following sterilization of the composition with radiation.